The invention relates to an affinity sensor for detecting specific molecular binding events, as is particularly used in the molecularbiological field, for example, in medical diagnostics, in biosensor technology or in DNA-microarray technology, and application of the same.
Biosensors are solid phase measuring devices that are comprised of at least one biological receptor, a transducer and a subsequently connected electronic unit.
The receptor utilizes biologically active reagents such as, for example, is antibodies for detecting a specific substance such as, for example, antigens. The transduction of detection events into detectable signals is performed by the transducer, for example, by electrochemical, optical, piezoelectric, or calorimetric methods. Thereby, the coupling of the detection events to the transducer can be carried out indirectly or directly. In the first case, the detection events modulate a process which is detected by the transducer. In the second case, the detection events themselves are recorded by the transducer. The transducer is connected to an electronic unit, for example, to a microprocessor followed by modules for signal detection and evaluation.
There are numerous application possibilities for such biosensors operating on the basis of molecular detection. These are, among others, in fields of detection and concentration analysis of biomolecules, kinetic and equilibrium analysis of biochemical reactions, control of fermentation processes, evaluation of receptor-cell-interactions, clinical analysis, and cell demotion.
The detection of the presence of bioactive molecules will be performed in the case of nucleic acids, for example, by hybridization with specific and marked nucleic acid probes. The marking of the probes is achieved by enzymatic inclusion of nucleotides that carry radioisotopes such as, for example, tritium, sulphur-35 or phosphorus-32, non-radioactive molecules such as, for example, digoxigenin or biotin and non-radioactive fluorescent molecules, respectively, such as, for example, fluoresceinisothiocyanat or 7-amino-4methylcumarin-3-acetate or metallic particles such as, for example, gold (Nicholl, D. S. T., 1995: Genetische Methoden, Spektrumis Akademischer Verlag Heidelberg, p. 24-27).
In the case of antigens, such as peptides or proteins, the detection of the presence of bioactive molecules is achieved by specific and marked antibodies. The marking of the antibodies is performed by coupling of radioisotopes such as, for example, iodine-125 or tritium, to tyrosine-residuals and histidine-residuals, respectively, by nonradioactive enzymes, for example, alkaline phosphatase or peroxidase, whereby the enzymatic activity is measured, for example, by the conversion of a colorless product into a colored one, by nonradioactive enzymes, for example, haematin which effects the chemiluminescent reaction of hydrogen peroxide and luminol, by nonradioactive enzymes, for example, luciferase which effects bioluminescence by means of phosphorized luciferin, or by metallic particles such as, for example, gold (Liddell, E. and Weeks, I., 1996: Antikoerpertechniken, Spektrum Akademischer Verlag Heidelberg, p. 87-107).
The signals from the various marker-molecules used will be evaluated by radio-chemical or electrochemical methods, by optical, piezoelectric, or calorimetric methods for indicating molecular detection events. Thereby, the size of the marker-molecules which emit single signals will lie in the nanometer area.
The optical and electrochemical methods for representing molecular binding events are the currently most utilized ones.
The problem of the various optical methods is, that the sensitivity and the spatial resolution of the signals emitted by the individual markermolecules is too low for many applications, that the binding between two links of a specific molecular binding pair cannot be detected, and that the signals are very often superimposed by an unspecific background. These problems of image generating methods can only be eliminated in part by an experimental amplification of the signal or by a computer aided statistical image analyzing method.
The technical limits of the current automation of the image analyzing on the basis of chip technology lies in a read-out of various microarray spots. Most of the available technologies are based on detection of fluorescence marked binding pairs, which are held in a specific manner to a surface of a chip, whereby the fluorescence detection is performed by an optical read-out of reactive centers of microarrays. The application of fluorescent or chemiluminescent samples is thereby utilized just as in the conventional method described hereinbefore and is combined with the CCD-imaging (Eggers, M. et al., 1996: Professional Program Proceedings, in Electro '96. IEEE, New York, N.Y., USA, 364 pp.; Heller, M. J., 1996: IEEE Engineering-in-Medicine-and-Biology-Magazine 15: 100-104), whereby also here the mentioned problems of the conventional image analyzing occur and a binding between two links of a specific molecular binding pair cannot be detected.
The detection of the presence of bioactive molecules can also be obtained by an electrochemical approach by various methods, apart from the commonly used optical methods.
The measurement of redox potency variations in biomolecules is a well-known possibility, which is accompanied by specific binding events, for example, on enzymes. Thereby, the redox potential variations are measured by way of a single electrode, which is provided with molecules, and a reference electrode (Heller, A., 1992: Electrical connection of enzyme redox centers to electrodes, J. Phys. Chem. 96: 3579-3587).
The disadvantage of this method lies in the fact that only one single electronic event occurs for one biomolecular binding event, whereby the variation of the redox state, which is effected, lasts only for a short time, so that the detection of each individual binding event had to take place flash-like. This is not possible. The signal obtained is only cumulative so that rare binding events cannot be detected by this as technology.
A further possibility for detecting the presence of bioactive molecules in an electrical way is to use biosensors in the form of special measuring electrodes. Such special measuring electrodes generally are comprised of a (strepto)-avidin coated electrode, whereby the (strepto)-avidin has the property to specifically bind biotin molecules. In this way it is possible to detect peptides, oligonucleotides, oligosaccharides and polysaccharides as well as lipides which are marked with biotin or biotin-derivatives, respectively to couple these as ligands to the (strepto)-avid-layer. In the latter case, the biotin molecules are the coupling elements. Generally, these biosensors allow detection of antibody/antigen binding pairs, antibody/partial antigen binding pairs, saccharide/lectin binding pairs, protein/nucleic acids binding pairs, and nucleinic acids/nucleinic acids binding pairs. The detection of the biochemical events occurring at the special measuring electrode takes place in a similar way to that of the before described technology based on redox system, namely, by measuring the potential variations across a single electrode compared to a reference electrode (Davis, et al., 1995: Element of biosensor construction; Enzyme Microb. Technol. 17: 130-1035).
A substantial disadvantage of this conventional biosensor technology is the inherent low sensitivity of the measurements at across the measuring electrodes that cannot be eliminated in that the ligands in an infinitely great density are bound to the measuring electrode, for example, by use of a dextran layer. Due to the additional deposition of, as for example, a dextran layer and due to the spatial arrangement of the ligands, the concentration of ligands on the electrodes is indeed raised up to the sixfold compared to a ligand single layer, but a detection of rare binding events or even of a binding between two elements of a special molecular binding pair is not possible.
Further known possibilities are:                the anchoring of specific antibodies on a semiconductor gate of a field-effect transistor, whereby a variation in the charge distribution and, hence, in the circuit of the field-effect transistor is obtained by the selective binding of antigens to the special antibody layer;        the immobilizing of special antibodies on the surface of an fiber, whereby measurable optical phenomena such as, for example, interfering waves and surface plasmons appear due to the selective binding of antigens to special antibody layers at the site of intersection between the fiber optics and the liquid;        as well as the method of surface plasmon resonance, in which, at a definite angle of incidence of light, the refractive index of a medium is, due to the selective coupling of antigens, measurably varied at a metal-coated glass body which is provided with specific antibodies (Liddell E. and Weeks, 1., 1996: Antikoerpertechniken, Spektrum Akademischer Verlag Heidelberg, p. 156-158).        
The disadvantage of these methods is that rare binding events cannot be detected by these technologies.
At present there are only a few methods available which allow a rapid detection of bindings between molecules at low concentrations or even with single molecule pairs (Lemieux, Bertrand et al., “Overview of DNA chip technology.” Molecular Breading 4: 277-289, 1988), though the biochemical process of the binding pair formation with biosensors, for example, the hybridization of two nucleotide strands or the binding of antibodies to antigens itself runs very quickly, that is, within the area of seconds; biochips can be provided with binding molecules, for example, with specific oligonucleotides (U.S. Pat. No. 5,445,934) or specific proteins (U.S. Pat. No. 5,077,210) so that a chip technology will be possible (Osborne, J. C., 1994: Genosensors. Conference Record of WESCON/94. Idea/Microelectronics. IEEE, New York, N.Y. USA: 434 pp.; Eggers, M. D. et al., 1993: Genosensors, microfabricated devices for automated DNA sequence analysis. Proc. SPIE-Int. Soc. Opt. Eng. 1998), by which the presence of definite biomolecules can be detected within a few minutes, for example, the presence of genes by use of specific oligonucleotide probes or antigens by use of specific antibodies, and by which great prod are indited in the field of biology or medicine, particularly as concerns genetic investigations (Chee, M. et al. 1996: Accessing genetic information with high-density DNA arrays. Science 274: 610-614).
A very promising approach as concerns the detection of binding events between nucleic acid bindings has recently been given by the utilization of the dielectric relaxation frequencies of the DNA to distinguish between hybridized and non-hybridized samples (Beattie et al. 1993. Clin Chem. 39: 719-722). The detection of the differences in frequencies, however, requires equipment which still is very expensive and which, moreover, still is far from being utilized as a matter of routine.
Furthermore, there is known another way to electronically distinguish hybridized samples from non-hybridized ones, which consists in determining the speed of the electron movements along the DNA strands (U.S. Pat. No. 5,780,234). This determination is based on the fact that the arrangement of the pi-electron orbits in the double-ended DNA causes the electrons to move faster in double-stranded DNA, that is, in hybridized DNA than in single-stranded DNA (Lipkin et al., 1995: Identifying DNA by the speed of electrons Science News 147, 117 pp.). To allow for a determination of these electron movements, the target has to be positioned exactly between two molecules. One of these molecules has to be chemically modified in such a way that it acts as an electron donor and the other one such, that it acts as an electron acceptor, so that there is a flow of electrons via electrodes measurable.
This expensive method has the disadvantage that it limits its application to the detection of single-sanded nucleic acids fragments of a defined length and that it is not suited for further biomolecules.
Furthermore, one of the methods for an electrical detection of particles is known from Bezryadin, A., Dekker, C., and Schmid G., 1997: “Electrostatic trapping single conducting nanoparticles between nanoelectrodes.” in Applied Physics Leers 71: 1273-1275, in which nanoparticles are captured in a gap formed by electrode in that a voltage is applied across the electrodes and the capturing of the particles is detected by way of the flow of the current. In contrast to the binding events of biomolecule pairs there is no specific biochemical binding of the nanoparticles, but the particle is bound to the electrode gap by the electric field.
There is also known from a work by Braun, E., Eichen, Y., Sivan, U., and Ben-Yoseph, G., 1998: “DNA templated assembly and electrode attachment of a conducting silver wire.” in Nature 391: 775-778, that DNA molecules can be held between two micro structurized electrodes and these molecule only exhibited an electric conductivity after having been silver coated, whereby this conductivity has nothing to do with specific biochemical binding events of biomolecule pairs.
Alivisatos, A. P., Johnson, K. P., Peng, X., Wilson, T. E., Loweth, C. J., Bruchez Jr. M., P. and Schulz, P., G., 1996: “Organization of nanocrystal molecules using DNA” in Nature 382: 609-611, generated complexes from short single-stringed DNA-molecules and their complementary single-stringed DNA-molecules marked with gold-particles in solution and deposited these on a TEM-grid with a carbon film for a characterization by electron microscope. An electric characterization, however, of the molecule pair binding did not take place.